Examination apparatus and focusing method of examination apparatus

ABSTRACT

A focusing method for an examination apparatus that can quickly and easily perform focusing for fluoroscopy is provided. The focusing method, for an examination apparatus that can observe fluorescence emitted from a specimen, includes a first step of irradiating the specimen with light via an objective lens to generate reflected light and fluorescence; a second step of performing focusing with respect to the surface of the specimen using the reflected light from the specimen; and a third step of performing focusing for the fluorescence based on the focal position of the specimen surface in the second step.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a focusing method for an examinationapparatus, such as a microscope, that can observe fluorescence emittedfrom a specimen.

2. Description of Related Art

In the related art, fluorescence microscopes are known examinationapparatuses. In fluorescence microscopes, excitation light from a lightsource is focused onto a specimen by an objective lens, fluorescenceemitted from the specimen passes through the objective lens and isfocused by an imaging lens, and the fluorescence is transmitted througha prism disposed inside a lens barrel to form an image.

In the fluoroscopy carried out with such a fluorescence microscope, thefollowing steps are performed: (a) first, the specimen is initiallyfocused; (b) next, the illumination intensity of the light source isreduced; (c) at the same time, the sensitivity of a photosensor isincreased; and (d) the focal position is finely adjusted. While viewingthe acquired images, steps (b) and (c) are repeated until a suitableimage is displayed, whereupon fluoroscopy can be carried out.

However, many operations are involved in such fluoroscopy, such asperforming coarse adjustment and fine adjustment of the focal positionwhile adjusting the illumination intensity. Therefore, this methodsuffers from the problem that an operator who is unaccustomed to suchoperations may easily make a mistake.

In addition, recently there have been more applications involvingexamination of protein-containing specimens (such as GFP). With suchprotein-containing specimens, however, the fluorescence intensity isvery low. Therefore, if the focusing in step (a) described above takestoo long or if the illumination intensity for quickly carrying out thefocusing in step (a) is increased too much, the level of fluorescencefrom the specimen decreases due to bleaching of the specimen. Thisresults in a problem in that fluoroscopy cannot be achieved.

On the other hand, scanning laser microscopes have recently beengathering attention. In such microscopes, laser light serving asexcitation light is two-dimensionally scanned onto a specimen,fluorescence generated in the specimen passes through a pinhole and isdetected in a detector, and the output of the detector is converted to adigital signal. Thereafter, the digital signal is stored in a memory andis displayed as an image. These scanning laser microscopes also have afocus adjusting function for adjusting the focal position of the image.

Japanese Unexamined Patent Application Publication No. HEI-9-218355discloses an example of a focus adjusting method for a scanning lasermicroscope. In this method, initially a pinhole is removed from thelight path and the focal position is roughly found, based onnon-confocal detection, with a photodetector. Then, the pinhole isinserted into the light path and confocal detection is performed withthe detector to more precisely find the focal position.

However, in the focusing method in Japanese Unexamined PatentApplication Publication No. HEI-9-218355, when using aprotein-containing specimen or the like whose fluorescence intensity isextremely low, it also takes a long time to roughly detect the focusposition with the non-confocal detection. In order to more quicklyperform detection of the focus position with non-confocal detection, thelaser beam intensity can be increased; however, if the laser beamintensity is increased too much, there is a problem in that thefluorescence becomes reduced due to bleaching of the specimen, whichmakes it difficult to perform fluoroscopy.

BRIEF SUMMARY OF THE INVENTION

In light of the problems described above, it is an object of the presentinvention to provide an examination apparatus and a focusing method foran examination apparatus in which focusing for fluoroscopy can beperformed quickly and easily.

In a first aspect, the present invention provides a focusing method foran examination apparatus that can observe fluorescence emitted from aspecimen, the focusing method including a first step of irradiating thespecimen with light via an objective lens to generate reflected lightand fluorescence; a second step of performing focusing with respect tothe surface of the specimen using the reflected light from the specimen;and a third step of performing focusing for the fluorescence based onthe focal position of the specimen surface detected in the second step.

In a second aspect, the present invention provides a focusing method foran examination apparatus according to the first aspect, wherein in thesecond step, the focal position for the surface of the specimen isdetected from the luminance of the reflected light from the specimen,and in the third step, focusing is performed for the fluorescence fromthe luminance of the fluorescence from the focal position detected inthe second step.

In a third aspect, the present invention provides a focusing method foran examination apparatus according to the second aspect, wherein in thethird step, a gain for detecting the luminance of the fluorescence fromthe focal position is adjusted so as to increase.

In a fourth aspect, the present invention provides a focusing method foran examination apparatus according to one of the first to third aspects,wherein in the third step, the focal position is moved in the inwarddirection of the specimen from the focal position detected in the secondstep and focusing for the fluorescence is performed.

In a fifth aspect, the present invention provides a focusing method foran examination apparatus that can observe fluorescence emitted from aspecimen, the focusing method including a first step of irradiating thespecimen with light via an objective lens to generate reflected lightand fluorescence; a second step of performing focusing with respect tothe surface of the specimen using the reflected light from the specimen;and a third step of performing examination for the fluorescence based onthe focal position of the specimen surface detected in the second step.

In a sixth aspect, the present invention provides a focusing method foran examination apparatus according to the fifth aspect, wherein in thefirst step, part of the specimen surface is irradiated with light togenerate the reflected light.

In a seventh aspect, the present invention provides a focusing methodfor an examination apparatus that can observe fluorescence emitted froma specimen, the focusing method including a first step of irradiatingthe specimen with light via an objective lens to generate reflectedlight and fluorescence; a second step of performing focusing withrespect to the surface of the specimen using an acquired reflected-lightimage of the specimen; and a third step of performing focusing for thefluorescence using an acquired fluorescence image based on the focalposition for the specimen surface detected in the second step.

In an eighth aspect, the present invention provides a focusing methodfor an examination apparatus according to the seventh aspect, wherein inthe third step, the focal position for the fluorescence examination iscorrected based on the chromatic aberration of the objective lens.

In a ninth aspect, the present invention provides a focusing method foran examination apparatus according to the seventh or eighth aspect,wherein in the second and third steps, acquired images are obtained bymeans of a mechanism producing a confocal effect.

In a tenth aspect, the present invention provides a focusing method foran examination apparatus according to one of the first to ninth aspects,wherein a variable focus lens having a focus-varying part is used as theobjective lens.

In an eleventh aspect, the present invention provides an examinationapparatus in which a focusing method according to one of the first totenth aspects is used.

With the present invention, since focusing is performed using reflectedlight from the surface of the specimen followed by focusing forfluorescence using this focal position as a basis, it is possible toquickly and easily perform fluoroscopy even if the level of fluorescenceemitted from the specimen is low, which normally makes focusingdifficult.

Also, with the present invention, since the operations up to focusingfor fluorescence can be performed quickly, it is possible to keep anydamage to the specimen, such as bleaching, to a minimum, which allowsstable and superior fluoroscopy to be realized.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 schematically shows the configuration of a scanning lasermicroscope in which a focusing method according to a first embodiment ofthe present invention is employed.

FIG. 2 is a flowchart for explaining the focusing method according tothe first embodiment.

FIG. 3 shows a modified example of a scanning laser microscope in whicha focusing method according to a first embodiment of the presentinvention is employed.

FIG. 4 schematically shows the configuration of a scanning lasermicroscope in which a focusing method according to a second embodimentof the present invention is employed.

FIG. 5 is a flowchart for explaining the focusing method according tothe second embodiment.

FIGS. 6A and 6B are diagrams for explaining the focusing methodaccording to the second embodiment.

FIG. 7 schematically shows the configuration of a confocal microscope inwhich a focusing method according to a third embodiment of the presentinvention is employed.

FIG. 8 is a flowchart for explaining the focusing method according tothe third embodiment,

FIG. 9 schematically shows a variable focus lens used in a fourthembodiment of the present invention.

FIG. 10 is a diagram for explaining the focusing method of the fourthembodiment.

FIG. 11 schematically shows an optical microscope in which a focusingmethod according to a fifth embodiment of the present invention isemployed.

FIG. 12 is a flowchart for explaining the focusing method according tothe fifth embodiment.

FIG. 13 is a diagram for explaining the focusing method of the fifthembodiment when a rigid-borescope objective lens is used.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be described below withreference to the drawings.

FIRST EMBODIMENT

FIG. 1 schematically shows the configuration of a scanning lasermicroscope in which a focusing method according to a first embodiment ofthe present invention is employed. In FIG. 1, reference numeral 1represents a laser light source, and this laser light source 1 generatesmultispectral laser light.

A laser modulating unit 2 is disposed in the optical path of the laserlight from the laser light source 1. The laser modulating unit 2 isformed, for example, of an AOTF (acousto-optic tunable filter), whichcan make the laser light from the laser light source 1 monochromatic andcan adjust the intensity thereof based on a modulation signal from asystem controller 14.

The laser light emitted from the laser modulating unit 2 is thenintroduced to an optical unit 3. In the optical unit 3, a cube 4 isdisposed in the light path of the laser light emitted from the lasermodulating unit 2. The cube 4 is mounted on a turret (not shown) and itcan be replaced with another cube having different characteristics,either with a motor or manually. In this case, a cube having ahalf-mirror characteristic with 20% reflectivity and 80% transmission,and a cube having a dichroic mirror for reflecting the laser light fromthe laser light source 1 and transmitting fluorescence from a specimen 7are used as the cube 4.

Galvano mirrors 5 are disposed in the reflected light path of the cube4. The galvano mirrors 5 include a pair of mirrors for deflecting lightin two orthogonal directions, thus scanning the laser lighttwo-dimensionally (in the X and Y axial directions) with these mirrors.

The laser light scanned with the galvano mirrors 5 is then introduced tothe microscope main body 19. In the microscope main body 19, a stage 11on which the specimen 7 is mounted is disposed in the light path of thelaser light scanned with the galvano mirrors 5, with an objective lens 6disposed therebetween. The stage 11 is moved upwards and downwards (inthe Z-axis direction) along the optical axis O of the objective lens 6by a motor 12 a that is controlled by a Z-axis controller 12. With thismovement, the specimen 7 can be brought into focus by changing therelative distance between the objective lens 6 and the specimen 7. Inthis case, the Z-axis controller 12 is controlled by a Z-position signalfrom the system controller 14. Also, the displacement of the stage 11 ismonitored by the Z-axis controller 12 and is sent back to the systemcontroller 14.

With this configuration, the light emitted from the galvano mirrors 5passes through the objective lens 6 and is focused onto the specimen 7on the stage 11. Also, detection light (reflected light or fluorescence)emitted from the specimen 7 returns to the cube 4 via the objective lens6 and the galvano mirrors 5.

A mirror 8 is disposed in the transmission light path of the cube 4. Abarrier filter 9, a pinhole 13, and a photosensor 10 are disposed in thereflection light path of the mirror 8.

In this case, two types of element are used for the barrier filter 9:one having a characteristic whereby reflected light from the specimen 7is transmitted, like the laser light from the laser light source 1, andanother having a characteristic whereby only fluorescence wavelengthsemitted from the specimen 7 are transmitted and other wavelengths arefiltered. These two types of barrier filter 9 are mounted on a turret(not shown) and are selectively inserted into the light path based on acontrol signal from the system controller 14. The pinhole 13 is disposedat a confocal position, which is an optically conjugate position withrespect to the focal point of the objective lens 6. The pinhole 13serves as a confocal detector for the detection light (reflected lightor fluorescence). The photosensor 10 detects the detection lightconfocally detected by the pinhole 13, and photoelectrically convertsthis detection light according to an adjustable gain signal from thesystem controller 14 to output an image luminance signal.

An image processing unit 15 is connected to the photosensor 10. Theimage processing unit 15 amplifies the image luminance signal from thephotosensor 10 with a certain gain and offset according to a controlsignal from the system controller 14 to generate image data, anddisplays this image data on a monitor 16.

Next, a description of the focusing operation of the scanning lasermicroscope with this configuration will be described with reference tothe flowchart shown in FIG. 2. First, in step 201, the optical elementsare set-up. In this case, the cube 4 that has a half-mirror with 20%reflectivity and 80% transmission is inserted in the light path, and thebarrier filter 9 having a characteristic whereby it transmits reflectedlight from the specimen 7 is also inserted in the optical path.

Next, in step 202, laser light is emitted from the laser light source 1.The laser light is attenuated by the half-mirror of the cube 4, istwo-dimensionally scanned by the galvano mirrors 5, is incident on theobjective lens 6, and is focused onto the specimen 7 on the stage 11.The light reflected at the surface of the specimen 7 returns to the cube4 via the objective lens 6 and the galvano mirrors 5.

The half-mirror of the cube 4 transmits most of the reflected light fromthe specimen 7. The light transmitted through the half-mirror of thecube 4 is then reflected at the mirror 8, passes through the barrierfilter 9, and is incident on the pinhole 13 where it is confocallydetected. The reflected light confocally detected by the pinhole 13 isthen received by the photosensor 10.

Next, in steps 203 and 204, focusing is carried out while adjusting thegain of the photosensor 10 and the image processing unit 15. In thiscase, the stage 11 is moved upwards and downwards (in the Z-axisdirection) with the motor 12 a by means of the Z-axis controller 12.With this movement of the stage 11, the intensity of the reflected lightfrom the specimen 7 changes, and the luminance of the image data fromthe image processing unit 15, which is based on the image luminancesignal from the photosensor 10, also changes. At this time, the systemcontroller 14 ensures that the luminance of the image data does notbecome saturated by adjusting the gain of the photosensor 10 and theimage processing unit 15.

Then, when the luminance of the image data from the image processingunit 15 is maximized, movement of the stage 11 is stopped. At this time,the position at which the stage 11 is stopped defines the focal positionof the surface of the specimen 7.

The reason why focusing is carried out using reflected light from thespecimen 7 is because the reflected light is brighter than thefluorescence, generally a few tens to a few hundred times brighter.

Next, in step 205, the optical elements are set-up again. In this case,the cube 4 that has a dichroic mirror for reflecting laser light fromthe laser light source 1 and for transmitting fluorescence from thespecimen 7 is inserted in the light path, and the barrier filter thathas a characteristic whereby it transmits fluorescence from the specimen7 is also inserted in the light path.

With this configuration, the fluorescence emitted from the specimen 8 inresponse to laser light excitation returns to the cube 4 via theobjective lens 6 and the galvano mirrors 5, is reflected at the mirror8, is transmitted through the barrier filter 9, and is incident on thepinhole 13 where it is confocally detected. The fluorescence confocallydetected at the pinhole 13 is then received by the photosensor 10.

Next, is steps 206 and 207, fine focusing adjustment is carried outbased on the fluorescence while adjusting the gain of the photosensor 10and the image processing unit 15. In this case, since the fluorescenceis less bright than the reflected light, gain adjustment is performed soas to increase the gain of the photosensor 10 and the image processingunit 15.

In this case, the stage 11 is moved with the motor 12 a by means of theZ-axis controller 12. Cells are often used as the specimen 7 influoroscopy, and the nucleus or strands in the cells are marked with afluorescent sample to carry out examination. Because of this, focusingof the interior of the specimen 7 is often carried out as opposed tofocusing of the surface of the specimen 7 with reflected light, asdescribed above.

Therefore, the stage 11, which is positioned at the focal position forthe surface of the specimen 7 based on reflected light, must be movedfurther towards the interior of the specimen 7. Then, the movement ofthe stage 11 should be stopped at the point where the luminance of theimage data from the image processing unit 15 is maximized. The positionof the stage 11 at this point then defines the focal position for thefluorescence emitted from the specimen 7.

Thereafter, fluoroscopy is carried out in step 208.

Accordingly, since focusing is initially performed using reflected lightfrom the surface of the specimen 7 followed by focusing for fluorescenceusing this focal position as a basis, it is possible to quickly andeasily perform focusing for fluoroscopy even when the intensity offluorescence emitted from the specimen 7 is low, which normally makesfocusing difficult.

Also, since focusing of the specimen 7 for fluoroscopy can be carriedout in a short time, any damage such as bleaching of the specimen iskept to a minimum, which allows stable, superior fluoroscopy to berealized.

Furthermore, since the fluorescence focusing operation is carried outafter focusing based on the reflected light and is performed onlytowards the interior of the specimen 7, no unnecessary operations arerequired up to the fluorescence focusing, which allows focusing to becarried out efficiently.

The above-described series of operations may be incorporated as afunction of the microscope. In this case, for example, by providing abutton on the GUI (graphical user interface) of an application, theoperator can press the button to automatically carry out theseoperations under program control.

As a scanning laser microscope in which a focusing method according tothe first embodiment of the present invention is employed, a scanninglaser microscope 72 as shown in FIG. 3 may be used which comprises asmall scan head 70 having an objective lens 6′ with a small diameter,the tip of which is to be brought close to or inserted into a smallexperimental animal, that is a specimen 7, and a support 71 whichsupports the scan head 70 in a manner such that the position of the scanhead 70 is adjustable. In this case, it is preferable that thephotosensor 10 be disposed outside the scan head 70 so as to facilitatehanding of the scan head 70. Reference numeral 73 represents a supportstand, reference numeral 74 represents a vertically movable member, andreference numeral 75 represents an angle adjusting member.

This scanning laser microscope 72 allows observation of the specimen 7such as a mouse in a living state by inserting the objecting lens 6′with a small diameter into the specimen 7 such as a mouse with a lowlevel of invasiveness. In this case, the specimen 7 such as a mouse canbe observed from a desired direction by raising or lowering thevertically movable member 74 with respect to the support stand 73 andadjusting the angle of the scan head 70 using the angle adjusting member75 so as to adjust the position of the tip of the objective lens 6′ andthe angle of the objective lens 6′.

SECOND EMBODIMENT

Next, a second embodiment of the present invention will be described.

FIG. 4 schematically shows the configuration of a scanning-lasermicroscope using a focusing method according to the second embodiment ofthe present invention. The same parts as shown in FIG. 1 are assignedthe same reference numerals.

In this case, a laser light source 21 is provided in addition to thelaser light source 1. Also, a half-mirror 22 is disposed in the lightpath of the laser light from the laser light source 1. Since thishalf-mirror transmits laser light from the laser light source 1 andreflects laser light from the laser light source 21, the laser beamsfrom the laser light sources 1 and 21 are combined onto the same lightpath.

The laser light source 1 is for generating reflected light at thespecimen 7. The laser light source 21 is for exciting fluorescence inthe specimen 7, and therefore, an optimum light source that matches theexcitation wavelength for exciting fluorescence in the specimen 7 isused. If a device that can output a plurality of wavelengths with asingle laser light source is used, only one of the laser light sources 1and 21 is required.

A laser modulating unit 23 is disposed in the emission light path of thehalf-mirror 22. This laser modulating unit 23 is formed of an AOTF(acousto-optic tunable filter), like that described above, which allowsthe intensity of the laser light from the laser light sources 1 and 21to be modulated. In addition, since the light modulation can be changedat a speed sufficiently higher than the image sampling speed, the lightmodulating unit 23 can be turned on and off to limit the scanning regionof the laser light from the laser light source 1. For example, eventhough the region scanned on the specimen by the galvano mirrors is arectangular raster-scanning region, it is possible to make the regionirradiated with laser light circular by suitably turning the lasermodulating unit 23 on and off.

A half-mirror 24 is disposed in the light path of the laser lightemitted from the laser modulating unit 23. This half mirror has areflectivity of 20% and a transmission of 80%.

A structure including the above-described galvano mirrors 5 is disposedin the reflection light path from the half-mirror 24, and a dichroicmirror 25 and a mirror 8 are disposed in the transmission light path.The dichroic mirror 25 has a characteristic whereby it reflects thelaser light from the laser light source 1 and transmits fluorescencefrom the specimen 7. A barrier filter 26, a pinhole 27, and aphotosensor 28 are disposed in the reflection light path of the dichroicmirror 25.

The barrier filter 26 transmits only the wavelength of the laser lightfrom the laser light source 1. The pinhole 27 is disposed at a confocalposition, which is an optically conjugate position with respect to thefocal point of the objective lens 6; therefore, the pinhole 17 serves toconfocally detect the reflected light. The photosensor 28 is fordetecting the reflected light confocally detected by the pinhole 27; thephotosensor 28 photoelectrically converts the detection light accordingto a variable gain signal from the system controller 14 to output animage luminance signal.

A barrier filter 9, a pinhole 13, and a photosensor 10, like those shownin FIG. 1, are disposed in the reflection light path of the mirror 8.The barrier filter 9 used here has a characteristic whereby it transmitsonly the fluorescence wavelength emitted by the specimen 7 and filtersother wavelengths.

The image processing unit 15 amplifies the image luminance signal fromthe photosensor 10 or 28 with a certain gain and offset, according to acontrol signal from the system controller 14, to produce image data, anddisplays this image data on the monitor 16.

The remaining configuration is the same as that shown in FIG. 1.

Next, the focusing operation of the scanning-laser microscope with thisconfiguration will be described with reference to the flowchart in FIG.5.

First, in step 401, the optical elements are set-up. In this case, anelement having 20% reflectivity and 80% transmission is used as thehalf-mirror 24; and element having a characteristic whereby it reflectsthe laser light from the laser light source 1 and transmits fluorescencefrom the specimen 7 is used as the dichroic mirror 25; an element thattransmits only the wavelength of the laser light from the laser lightsource 1 is used as the barrier filter 26; and an element that transmitsonly the fluorescence wavelength emitted by the specimen 7 and thatfilters the other wavelengths is used as the barrier filter 9.

Next, in step 402, the scanning region on the specimen 7 of the laserlight from the laser light source 1 is set using the laser modulatingunit 23. In this case, the scanning region set with the laser modulatingunit 23 can be arbitrarily selected by the user; if only a part of thespecimen 7 is to be scanned, only a part of the scanning region rasterscanned by the galvano mirrors 5 is selected, for example, the edges orcenter of the scanning region.

Next, in step 403, laser light is generated by the laser light sources 1and 21. The laser light from the laser light sources 1 and 21 isreflected at the half-mirror 24, is incident on the objective lens 6 viathe galvano mirrors 5, and is focused onto the specimen 7 on the stage11. In this case, the laser light from the laser light source 21irradiates the scanning region on the specimen 7 according to the rasterscanning of the galvano mirrors 5, whereas the laser light from thelaser light source 1 illuminates only a part of the scanning regionspecified by the laser modulating unit 23.

The fluorescence and reflected light coming from the specimen 7 returnto the half-mirror 24 via the objective lens 6 and the galvano mirrors5, and then pass through the half-mirror 24 to be incident on thedichroic mirror 25. The reflected light from the specimen 7 is reflectedat the dichroic mirror 25, is transmitted through the barrier filter 26,and is incident on the pinhole 27 to be confocally detected thereat. Thereflected light confocally detected at the pinhole 27 is then receivedat the photosensor 28. On the other hand, the fluorescence from thespecimen 7 is transmitted through the dichroic mirror 25, is reflectedat the mirror 8, is transmitted through the barrier filter 9, and isincident on the pinhole 13 to be confocally detected thereat. The lightconfocally detected at the pinhole 13 is then received at thephotosensor 10.

Next, in steps 404 and 405, fluoroscopy can be carried out at the sametime as adjusting the gain of the photosensor 28 and the imageprocessing unit 15 and performing focusing. In this case, to performfocusing, the stage 11 is moved upwards and downwards (in the Z-axisdirection) with the motor 12 a by means of the Z-axis controller 12, tofind the position where the luminance of the image data in the imageprocessing unit 15 is maximized, and this position defines the focalposition. At this time, the system controller 14 ensures that theluminance of the image date (reflection image) does not become saturatedby adjusting the gain of the photosensor 28 and the image processingunit 15.

On the other hand, the fluorescence received at the photosensor 10 isphotoelectrically converted and is input to the image processing unit 15as an image luminance signal. The image processing unit 15 amplifies theimage luminance signal from the photosensor 10 with a certain gain andoffset based on a control signal from the system controller 14 togenerate image data, and displays this image data on the monitor 16 as afluorescence image.

Accordingly, with this configuration, focusing is carried out using thereflected light from the surface of the specimen 7, and a fluorescenceimage can be examined while viewing the image at this focal point. As aresult, when carrying out in-vivo examination using a living organism ofan experimental animal as the specimen, as well as in in-vitroexamination, even if the specimen moves during examination and causesthe image to become out of focus, it is possible to quickly refocususing the reflection image, which allows examination to be carried outefficiently. These operations can be carried out by the user or they canbe carried out automatically under the control of the system controller14.

Also, since the region from which reflected light is obtained from thespecimen 7 can be restricted to a plane or point forming part of thescanning region, it is possible to keep any damage to the specimen, suchas bleaching, to a minimum, which enables superior fluoroscopy to berealized.

Separate light sources, that is, the laser light source 1 for reflectedlight and the laser light source 21 for excitation, are used in theembodiment described above; however, if reflection images andfluorescence images can be suitably acquired with a single laser lightsource, it is possible to obtain both reflection images and fluorescenceimages with the laser light source 1 or 21.

In the description given above, the reflected light from the specimen 7is transmitted through the barrier filter 26 and is incident on thepinhole 27; however, the pinhole 27 for reflected light may be set to ahave a large diameter or a small diameter, depending on the application.These diameters are set by a program provided in the system controller14 in advance, and can be automatically selected.

FIGS. 6A and 6B show the relationship between the focal position and theimage luminance for cases where the specimen 7 and the objective lens 6are used confocally and non-confocally. First, when the pinhole diameterof the pinhole 27 is set large, no confocal effect occurs and thereforea non-confocal mode is used. In this mode, when the focal position isclose to the surface of the specimen 7, the image luminance relationshipis shown by curve A in FIG. 6B, and a blurred image is observed. In sucha case, the surface of the specimen 7 is generally brought into focususing an autofocus technique. On the other hand, when the pinholediameter of the pinhole 27 is set small, the relationship between focalposition and image luminance of the specimen 7 is as shown by curve B inFIG. 6B. In this case, when fluorescence is produced at a cell insidethe specimen 7, the luminance is low at a focal position outside thespecimen 7 and rapidly rises as the focal position moves inside thespecimen 7. Therefore, when the stage 11 is moved so as to bring thespecimen 7 closer to the objective lens 6, if the focal position is setto a position where the image luminance exceeds a predeterminedthreshold value C, focusing can be easily carried out. Therefore, in thecase of a living organism in which the surface or interior of thespecimen 7 is to be examined, the focusing method using the confocalpoint is advantageous

THIRD EMBODIMENT

Next, a third embodiment of the present invention will be described.

FIG. 7 schematically shows the configuration of a confocal microscope inwhich a focusing method according to the third embodiment of the presentinvention is employed.

In this case, instead of the laser light source 1, a halogen lamp 31 isprovided. The halogen lamp 31 introduces white light into the opticalunit 3.

A cube 32 is disposed in the light path of the white light from thehalogen lamp 31. The cube 32 is mounted on a turret (not shown) and canbe replaced, either with a motor or manually, with another cube havingdifferent characteristics. In this case, a cube having a half-mirrorwith 20% reflectivity and 80% transmission and cube having a dichroicmirror that reflects light from the halogen lamp 31 and transmitsfluorescence from the specimen 7 are used as the cube 32.

A rotating disk 33 is disposed in the reflection light path of the cube32. The rotating disk 33 has slits formed in the circular disk surface,which allows a confocal effect to be obtained when the disk is rotatedto transmit light.

The light passing through the rotating disk 33 is introduced into themicroscope main body 19. In the microscope main body 19, the stage 11 onwhich the specimen 7 is mounted is disposed in the light path of thelight passing through the rotating disk 33, with the objective lens 6being disposed therebetween.

On the other hand a CCD camera 34 is disposed in the transmission lightpath of the cube 32. The CCD camera 34 acquires images of the detectionlight (reflected light or fluorescence) from the specimen 7, and outputsa reflection image signal or a fluorescence image signal based on acontrol signal from the system controller 14.

The image processing unit 15 is connected to the CCD camera 34. Theimage processing unit 15 amplifies the reflection image signal or thefluorescence image signal from the CCD camera 34 with a certain gain oroffset based on a control signal from the system controller 14 togenerate image data, and displays this image data on the monitor 16.

The system controller 14 can perform Z-axis control of the stage 11 onwhich the specimen 7 is mounted by means of a Z-axis controller 12,based on the reflection image signal or the fluorescence image signal.For example, the system controller 14 is capable of performingprocessing for setting the Z-axis position of the stage 11 at a pointwhere the luminance of the reflection image signal is maximized.

Next, a focusing operation of the confocal microscope with thisconfiguration will be described with reference to the flowchart shown inFIG. 8. First, in step 701, the optical elements are set-up. In thiscase, the cube 32 having the half-mirror with 20% reflectivity and 80%transmission is used.

Next, in step 702, the rotating disk 33 is rotated and light isirradiated from the halogen lamp 31. The light from the halogen lamp 31is attenuated at the half mirror in the cube 32 and is introduced to theobjective lens 6 via the rotating disk 33 to be focused onto thespecimen 7 on the stage 11. The light reflected at the surface of thespecimen 7 passes through the objective lens 6 and is incident on therotating disk 33, where a confocal effect occurs. The reflected light isthen transmitted by the half-mirror in the cube 32 and is imaged at theCCD camera 34.

Next, in steps 703 and 704, focusing is carried out while adjusting thegain and so on of the image processing unit 15. In this case, thereflection image acquired by the CCD camera 34 is transmitted as areflection image signal to the system controller 14 via the imageprocessing unit 15. The system controller 14 moves the stage 11 upwardsand downwards (in the Z-axis direction) by means of the Z-axiscontroller 12, according to the reflection image signal, and finds apoint where the luminance of the reflection image signal is maximized toset the focus position. At this time, the gain of the image from theimage processing unit 15 is adjusted if necessary.

Next, in step 705, the optical elements are set-up again. In this case,the cube 32 having the dichroic mirror that reflects light from thehalogen lamp 31 and that transmits fluorescence from the specimen 7 isused.

In this state, the fluorescence emitted from the specimen 7 in responseto excitation with light from the halogen lamp 31 passes through theobjective lens 6, is incident on the rotating disk 33, where a confocaleffect occurs, and returns to the cube 32. The fluorescence is thentransmitted through the dichroic mirror in the cube 32 and is imaged atthe CCD camera 34.

The fluorescence image acquired by the CCD camera 34 is then sent to theimage processing unit 15 as a fluorescence image signal. The imageprocessing unit 15 amplifies the fluorescence image signal from the CCDcamera 34 with a certain gain and offset, according to a control signalfrom the system controller 14, to generate image data, and displays theimage data on the monitor 16 (step 706). By doing so, a focusedfluorescence image that is not affected by the brightness of thefluorescent specimen is displayed on the monitor 16.

The focal planes of the reflection image and the fluorescence imageacquired in this way may differ depending on the amount of the chromaticaberration of the objective lens 6. In this case, in step 707, thesystem controller 14 calculates in advance the difference in focal planepositions due to the chromatic aberration, calculates the position ofthe focal plane of the fluorescence image from the position of the focalplane at which the reflection image is focused, and performs correctionby moving the stage 11 upwards and downwards (in the Z-axis direction)by means of the Z-axis controller 12.

Thereafter, in steps 708 and 709, the focal position thus obtained isrecorded, and then fluoroscopy is carried out.

Therefore, the procedure up to fluoroscopy can be carried out quicklyand in a straightforward manner even when the intensity of thefluorescence emitted from the specimen 7 is low, which normally makefocusing difficult.

Focusing of the reflection image is automatically performed based on theluminance of the image acquired by the CCD camera 34, and focusing ofthe fluorescence image is carried out by calculating the chromaticaberration, which allows the focusing to be carried out with almost nointervention by the user. Also, since a confocal effect is obtained byusing the rotating disk 33, more precise focusing can be achieved.

When defocusing occurs during fluoroscopy, it is possible to refocusagain using the reflected light simply by changing the cube 32 to theone having the half-mirror. Also, since the CCD camera 34 is used, ahigher frame rate is possible compared to a scanning-laser microscope,and it is also possible to carry out focusing quickly.

FOURTH EMBODIMENT

Next, a fourth embodiment of the present invention will be described.

In the above-described embodiments, the stage 11 on which the specimen 7is mounted is moved upwards and downwards; instead of this, however, avariable focus lens may be used. FIG. 9, schematically shows a variablefocus lens. A focus-varying part 42 formed of a liquid crystal lens, aliquid lens, or the like is provided in the tip of an objective lens 41to constitute a variable focus lens 40.

With this variable focus lens 40, when a control signal from a controlunit (not shown) is applied to the focus-varying unit 42, a pre-controlfocal plane D can be moved to a post-control focal plane E.

Since the focusing speed is much higher by using this variable focuslens 40 compared to moving the stage 11, the focusing operation can beperformed more quickly. Also, as shown in FIG. 10, when cells 7 a insidethe specimen 7 are to be examined, bringing the objective lens 6 closeto the specimen 7 after focusing at the surface of the specimen 7 withthe above-described objective lens 6 involves a risk that the lens 6will collide with the specimen 7. However, by using the variable focuslens 40, the variable focus lens 40 itself is not moved, and therefore,it is possible to examine the cell 7 a inside the specimen 7 simply bymoving the focal plane from D to E. Furthermore, using such a variablefocus lens 40 as the objective lens 6 provides an advantage in thatchromatic aberration mentioned in the fourth embodiment can becorrected.

FIFTH EMBODIMENT

Next, a fifth embodiment of the present invention will be described.

FIG. 11 schematically shows the configuration of an optical microscopein which a focusing method according to the fifth embodiment of thepresent invention is employed.

In FIG. 12, reference numeral 51 represents a microscope main body. Astage 53 on which a specimen 52 is mounted and an objective lens 54 aredisposed in this microscope main body 51 so as to face each other. Thestage 53 can be moved upwards and downwards (in the Z-axis direction)along the optical axis O of the objective lens 54 by turning a focusingknob 55. By changing the relative distance between the objective lens 54and the specimen 52 with this movement, the specimen 52 can be broughtinto focus.

A lamp 56 serving as a light source is provided on the microscope mainbody 51. The lamp 56 emits white light. A cube 57 is disposed in thelight path of the white light from the lamp 56. The cube 57 is providedinside the microscope main body 51 and can be replaced, either with amotor or manually, with another cube having different characteristics.In this case, a cube having a half-mirror with 20% reflectivity and 80%transmission and a cube having a dichroic mirror that reflects the whitelight from the lamp 56 and transmits detection light (reflection lightor fluorescence) from the specimen 52 are used as the cube 57, and thesecan be exchanged.

A lens barrel 58 is disposed in the transmission light path of the cube57. The lens barrel 58 allows visual observation of the detection lightfrom the specimen 52, which is transmitted through the cube 57.

Next, the focusing operation of the optical microscope having thisconfiguration will be described with reference to the flowchart shown inFIG. 12. First, in step 1101, the optical elements are set-up, namely,the cube 57 with the half-mirror is installed.

Next, in steps 1102 and 1103, the light from the lamp 56 is attenuatedat the cube 57, is incident on the objective lens 54, and is focusedonto the specimen 52 on the stage 53. The light reflected at the surfaceof the specimen 52 passes through the objective lens 54 and reaches thecube 57. The reflected light then passes through the cube 57 and isobserved as a reflection image using the lens barrel 58.

Next, in step 1104, the stage 53 is moved upwards and downwards (in theZ-axis direction) by turning the focusing knob 55, to bring the specimen52 into focus.

In step 1105, the optical elements are set-up again, this time toinstall the cube 57 with the dichroic mirror.

In this state, when light is radiated from the lamp 56, fluorescenceexcited in the specimen 52 reaches the cube 57 via the objective lens54, is transmitted through the cube 57, and is observed as afluorescence image with the lens barrel 58 (step 1106).

Therefore, the procedure up to fluoroscopy can be carried out quicklyand in a straightforward manner even when the intensity of thefluorescence emitted from the specimen 52 is low, which normally makefocusing difficult. Also, when a high-magnification lens is used as theobjective lens 54, even if defocusing occurs, it is possible to easilyrecreate the original fluoroscopy state by performing focusing using thereflected light from the specimen 52.

This concept can be applied to a microscope apparatus in which arigid-borescope objective lens is used for examining the internal organsof a living organism. FIG. 13 is an example using a rigid-borescopeobjective lens 61 of the type that is pressed against the surface 521 ofan internal organ instead of the objective lens 54. As shown in (a) inFIG. 13, when the rigid-borescope objective lens 61 is separated fromthe internal organ surface 521, the internal organ surface 521 isbrought into focus using the reflected light from the internal organsurface 521. Next, as shown in (b) in the same figure, a cell 521 a,inside the internal organ surface 521, that emits fluorescence isbrought into focus. In this case, when the cell 521 a that emitsfluorescence is located deeper below the internal organ surface 521, asshown in (c) in the same figure, the rigid-borescope objective lens 61is inserted inside the internal organ surface 51, and in this state, thecell 521 a that emits fluorescence is brought into focus. Naturally, thefluorescence inside the internal organ surface 521 may also be focusedby placing the tip of the rigid-borescope objective lens 61 against theinternal organ surface 521. Organ examination may be carried out whenthe organ is removed from the living organism. Also, in in-vivoexamination, the rigid-borescope objective lens 61 may be inserted intoan experimental animal to approach the organ to be examined.

In this examination apparatus, when using high-magnification lenses inthe rigid-borescope objective lens 61, even if there is some defocus dueto slight movement of the specimen 52, it is possible to easily returnto the original fluoroscopy state by performing focusing using theinitially reflected light from the specimen 52.

The present invention is not limited to the embodiments described above.Various modifications can be made without departing from the scope ofthe invention.

Furthermore, the embodiments described above include various aspects ofthe invention, and various aspects of the invention can be obtained bysuitably combining the plurality of disclosed structural elements. Forexample, even when various structural elements are removed from thecomplete structure disclosed in the embodiments, so long as the problemsdescribed above in the Summary of the Invention can be overcome and theadvantages described therein can be obtained, the configuration fromwhich these structural elements are removed can be considered as theinvention.

1. A focusing method for an examination apparatus that can observefluorescence emitted from a specimen, the focusing method comprising: afirst step of irradiating the specimen with light via an objective lensto generate reflected light and fluorescence; a second step ofperforming focusing with respect to the surface of the specimen usingthe reflected light from the specimen; and a third step of performingfocusing for the fluorescence based on the focal position of thespecimen surface detected in the second step; wherein in the secondstep, the focal position for the surface of the specimen is detectedfrom the luminance of the reflected light from the specimen, and in thethird step, focusing is performed for the fluorescence from theluminance of the fluorescence from the focal position detected in thesecond step.
 2. A focusing method for an examination apparatus accordingto claim 1, wherein in the third step, a gain for detecting theluminance of the fluorescence from the focal position is adjusted so asto increase.
 3. A focusing method for an examination apparatus accordingto claim 1, wherein a variable focus lens having a focus-varying part isused as the objective lens.
 4. An examination apparatus using thefocusing method according to claim
 1. 5. A focusing method for anexamination apparatus that can observe fluorescence emitted from aspecimen, the focusing method comprising: a first step of irradiatingthe specimen with light via an objective lens to generate reflectedlight and fluorescence; a second step of performing focusing withrespect to the surface of the specimen using the reflected light fromthe specimen; and a third step of performing focusing for thefluorescence based on the focal position of the specimen surfacedetected in the second step, wherein in the third step, the focalposition is moved in the inward direction of the specimen from the focalposition detected in the second step and focusing for the fluorescenceis performed.
 6. A focusing method for an examination apparatusaccording to claim 5, wherein a variable focus lens having afocus-varying part is used as the objective lens.
 7. An examinationapparatus using the focusing method according to claim
 5. 8. A focusingmethod for an examination apparatus that can observe fluorescenceemitted from an internal organ of a living organism, the focusing methodcomprising: bringing an objective lens of the examination apparatusclose to the internal organ; a first step of irradiating the internalorgan with light via the objective lens to generate reflected light andfluorescence; a second step of performing focusing with respect to thesurface of the internal organ using the reflected light from theinternal organ; and a third step of performing focusing for thefluorescence based on the focal position of the surface of the internalorgan detected in the second step, wherein in the second step, the focalposition for the surface of the internal organ is detected from theluminance of the reflected light from the internal organ, and in thethird step, focusing is performed for the fluorescence from theluminance of the fluorescence from the focal position detected in thesecond step.
 9. A focusing method for an examination apparatus accordingto claim 8, wherein a variable focus lens having a focus-varying part isused as the objective lens.
 10. An examination apparatus using thefocusing method according to claim
 8. 11. A focusing method for anexamination apparatus that can observe fluorescence emitted from aninternal organ of a living organism, the focusing method comprising:bringing an objective lens of the examination apparatus close to theinternal organ; a first step of irradiating the internal organ withlight via the objective lens to generate reflected light andfluorescence; a second step of performing focusing with respect to thesurface of the internal organ using the reflected light from theinternal organ; and a third step of performing focusing for thefluorescence based on the focal position of the surface of the internalorgan detected in the second step, wherein in the third step, the focalposition is moved in the inward direction of the internal organ from thefocal position detected in the second step and focusing for thefluorescence is performed.
 12. A focusing method for an examinationapparatus according to claim 11, wherein a variable focus lens having afocus-varying part is used as the objective lens.
 13. An examinationapparatus using the focusing method according to claim 11.